The endoribonuclease Dicer produces two types of small regulatory RNAs that regulate gene expression: small interfering RNAs (siRNAs) and microRNAs (miRNAs) (Bernstein et al., 2001; Grishok et al., 2001; Hutvágner et al., 2001; Ketting et al., 2001; Knight and Bass, 2001). In animals, siRNAs direct target mRNA cleavage (Elbashir et al., 2001c; Elbashir et al., 2001d), whereas miRNAs block target mRNA translation (Lee et al., 1993; Reinhart et al., 2000; Brennecke et al., 2003; Xu et al., 2003). Recent data suggest that both siRNAs and miRNAs incorporate into similar perhaps even identical protein complexes, and that a critical determinant of mRNA destruction versus translation regulation is the degree of sequence complementary between the small RNA and its mRNA target (Hutvágner and Zamore, 2002; Mourelatos et al., 2002; Zeng et al., 2002; Doench et al., 2003; Saxena et al., 2003; Zeng et al., 2003a).
Target RNA cleavage directed by siRNA is called RNA interference (RNAi). RNAi is a powerful method for the study of gene function in animals and plants and is being developed as a therapy for treating genetic disorders and viral infections. Biochemical studies in Drosophila S2 cells (Bernstein et al., 2001; Hammond et al., 2001 a; Caudy et al., 2002; Liu et al., 2003) and affinity purification (Martinez et al., 2002) or immunoprecipitation (Hutvágner and Zamore, 2002) from cultured human HeLa cells have identified protein components of the RNAi effector complex, the RNA-induced silencing complex (RISC; the RISC complex also functions in miRNA-mediated translational silencing). Genetic mutations that disrupt RNAi in C. elegans, Drosophila, green algae, fungi and plants have likewise identified proteins required for RNAi (Cogoni and Macino, 1997; Cogoni and Macino, 1999a; Cogoni and Macino, 1999b; Ketting et al., 1999; Tabara et al., 1999; Catalanotto et al., 2000; Dalmay et al., 2000; Fagard et al., 2000; Grishok et al., 2000; Ketting and Plasterk, 2000; Mourrain et al., 2000; Wu-Scharf et al., 2000; Dalmay et al., 2001; Catalanotto et al., 2002; Grishok and Mello, 2002; Tabara et al., 2002; Tijsterman et al., 2002a; Tijsterman et al., 2002b). Key steps in the RNAi pathway have also emerged from studies of RNAi reconstituted in cell-free extracts (Tuschl et al., 1999; Zamore et al., 2000; Hammond et al., 2001b; Nykänen et al., 2001; Martinez et al., 2002; Schwarz et al., 2002; Tang et al., 2003).
Recently hundreds of miRNAs have been identified in animals and plants (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001; Lagos-Quintana et al., 2002; Reinhart et al., 2002; Ambros et al., 2003; Aravin et al., 2003; Brennecke and Cohen, 2003; Lim et al., 2003). Of these, the biological functions of at least four animal miRNAs are known. In C. elegans, the miRNAs lin-4 (Locus link ID 266860; Lee et al., 1993; Olsen and Ambros, 1999) and let-7 (Locus link ID 266954; Reinhart et al., 2000) regulate developmental timing, whereas the Drosophila miRNAs bantam (Locus link ID 117376) and miR-14 (Locus link ID 170868) control cell survival by repressing translation of pro-apoptotic genes (Brennecke et al., 2003; Xu et al., 2003). Computational approaches have also been described to assist in identifying the mRNA targets of other miRNAs (Enright et al., 2003; Lewis et al., 2003; Stark et al., 2003). Despite the widespread use of RNAi to ‘knock down’ gene function and the increasing body of evidence supporting a role for miRNAs in RNA silencing, the mechanisms by which these processes occur are not yet fully understood. Accordingly, there exists a need for a more complete understanding of the mechanisms underlying RNA silencing (e.g., RNAi, miRNA-mediated translational silencing), as well as for compounds which can regulate RNA silencing.